Small Peptide Modulation of FGFR3-dependent Postnatal Lymphangiogenesis
David Perrault, BS, Gene K. Lee, MD, Sun Young Park, MS, Sunju Lee, PhD, Dongwon Choi, PhD, Eunson Jung, MS, Young Jin Seong, MS, Cynthia Sung, BS, Roy Yu, BS, Soo Jung Kim, ., Young-Kwon Hong, PhD, Alex K. Wong, MD.
Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA.
PURPOSE: The fibroblast growth factor receptor (FGFR) family is a group of highly conserved transmembrane receptors that are involved in a wide range normal developmental and post-developmental biologic processes as well as a wide range of human disease. Specifically regarding post-developmental lymphatic regeneration, FGFR3 has been implicated in the mechanism by which 9-cis retinoic acid (9-cisRA) pharmacologically induces lymphangiogenesis and improves lymphedema. The purpose of this study was to validate the efficacy of a novel small peptide FGFR3 inhibitor, named peptide P3, and to elucidate the role of FGFR3 in 9-cisRA induced lymphangiogenesis in vitro and in vivo with the aid of this peptide.
METHODS: Peptide P3 (P3) was identified as a specific inhibitor of FGFR3, synthesized, and validated for effective inhibition of FGFR3 and downstream signaling of MAPK (ERK1/2) in human lymphatic endothelial cells (LEC). A scrambled peptide (Sc) of random sequence was also synthesized as a peptide control. LEC proliferation, migration, and tubule formation were assessed via standard WST-1, confluent monolayer scratch, and two-dimensional Matrigel assays. Treatments groups included ethanol (vehicle control for 9-cisRA), ethanol+Sc, ethanol+P3, 9-cisRA, 9-cisRA+Sc, and 9-cisRA+P3. In vivo, the effect of peptide P3-mediated FGFR3 inhibition on 9-cisRA induced lymphangiogenesis was via tracheal lymphangiogenesis in a lymphatic reporter mouse (Prox1-GFP).
RESULTS: Peptide P3 effectively inhibited FGFR3 phosphorylation as well as downstream signaling of MAPK (ERK1/2). In vitro, peptide P3-mediated FGFR3 inhibition did not decrease LEC proliferation, migration, or tubule formation. However, peptide P3-mediated FGFR3 inhibition did block 9-cisRA stimulated LEC proliferation, migration, and tubule formation. In vivo, peptide P3-mediated FGFR3 inhibition was sufficient to inhibit 9-cisRA induced tracheal lymphangiogenesis.
CONCLUSION: FGFR3 does not appear to be essential to non-promoted LEC proliferation, migration, and tubule formation. However, FGFR3 may play a key role in LEC proliferation, migration, tubule formation, and post-natal in vivo lymphangiogenesis when pharmacologically induced by 9-cisRA. Since P3 has now been validated in multiple cell types and animal models, it has the potential to be used as a precise regulatory control element for 9-cisRA mediated lymphangiogenesis. In addition, given the role of FGFR3 in various malignancies and the specificity of P3 for FGFR3, P3 may have a role in treating cancer cells that are highly dependent on FGFR3 signaling.
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